The cholesterol-dependent cytolysins (CDCs) are a large family of related bacterial toxins produced by over 20 species of Gram-positive pathogenic bacteria. Several CDCs have been shown to contribute to the pathogenic mechanisms of these bacterial species. The CDCs are produced as soluble proteins that oligomerize into large pore-forming complexes on the surface of cholesterol-containing membranes. The presence of membrane cholesterol is required for the CDC pore-formation and is a hallmark of the CDCs. For over three decades the receptor for the CDCs was generally accepted as cholesterol but recent studies show that it is necessary for the prepore to pore conversion of the CDCs, an event that is downstream of membrane binding. Therefore, the longstanding dogma that the CDCs use cholesterol as their receptor appears less plausible. We have now determined that at least one member of the CDC family, intermedilysin (ILY) from Streptococcus intermedius, utilizes the GPI-anchored human CD59 (hCD59) as its receptor. Based on preliminary studies we hypothesize that 1) ILY domain 4 contains the residues required for binding to and specificity for hCD59, 2) the region and residues of hCD59 recognized by human complement proteins C8 and C9 also comprises the hCD59 binding site for ILY, and 3) ILY disengages from its receptor prior to pore formation. This proposal is designed to examine the interaction of ILY with hCD59 and to map out the structural domains of both ILY and hCD59 that are involved in this ligand-receptor interaction. The interaction of ILY with hCD59 will also be examined at various stages of the cytolytic mechanism to determine if the toxin disengages from receptor during the assembly of its pore complex. These hypotheses will be addressed in three specific aims that will establish the location of the binding sites on both hCD59 and ILY and will determine whether ILY undocks from receptor during the assembly of the pore-forming complex. The results of these studies will provide a basis for investigations of a new and significant aspect of the CDCs previously unavailable to the study of these toxins. The discovery and study of a CDC receptor will have a significant impact on the study of CDCs and will alter our perception of their role in a large number of diseases caused by a wide variety of Gram-positive pathogens.